Field of the Invention
The present invention relates generally to methods for genetically altering higher plant materials and reproducing whole plants therefrom. More particularly, the invention relates to a method for genetically transforming and regenerating pepper plants. The result of transformation coupled with regeneration is a pepper line or plant containing introduced DNA capable of expression. The invention further comprises an improved method of regenerating pepper (without transformation). Regeneration without transformation can be used for propagation or to produce variants of pepper, e.g., somaclonal variants, with a new characteristic. The new characteristic may be polyploidy (e.g., a tetraploid pepper).
Pepper is of the genus Capsicum, which is of the family Solanaceae, subfamily Solanoideae and tribe Solaneae. This genus includes the species Capsicum annuum and Capsicum frutescens. Peppers are cultivated and used around the world as sweet peppers such as the bell pepper; or as pungent chili peppers, jalapeno peppers, and Tabasco peppers (used to make Tabasco sauce); or as a source of dried powders of various colors such as paprika.
The types of cultivated peppers can be differentiated by pungency, fruit shape, and size. Non-pungent peppers used for the fresh market include the large, blocky, thick-fleshed Bell or Stuffing type (e.g., cv.s (cultivars) California Wonder, Yolo Wonder, Keystone Giant and Dulce Italians) and the medium-sized, heart-shaped, thick-fleshed Pimiento type (e.g., cv.s Pimiento, Pimiento Select, Pimiento Perfection, and Super Red Pimiento) peppers, and the long, blunt-ended, thin fleshed Cuban type (e.g., cv.s Cubanelle, and Aconcagua). Mildly pungent peppers used for the fresh market and for processing include the long, heart-shaped, thin-fleshed Ancho type (e.g., cv.s Mexican Chili, Ancho, and Mulato), and the long, blunt-ended, thin-fleshed Tuscan type (e.g., cv. Pepperoncini) peppers. The slightly more pungent Anaheim Chili (e.g., cv.s Anaheim Chili, Sandia, California Chili, Mild California, and New Mexican Chili) which is used mainly for processing has an elongate fruit which tapers to a point and medium flesh thickness. Pungent peppers used in both the fresh market and for processing include the long, cylindrical-thick fleshed Jalapeno (e.g., cv.s Jalapeno and Mild Jalapeno), the small, slender, tapering Serrano (e.g., cv. Serrano), and the irregularly shaped, thin-fleshed Cayenne (e.g., cv.s Cayenne Long Thick, Cayenne Long Slim, and Cayenne Long Red) peppers. In addition to the above C. annuum types, there are various C. frutescens type peppers (e.g., cv. Tabasco).
A type of pepper has been developed which does not fit into the above categorizations. It is a pepper with no pungency, but with a long, cylindrical (Jalapeno type) shape. Pepper lines of this type are called VEGISWEET (registered trademark), e.g., Vegisweet line 89288-2 and Vegisweet line 89300-1. These pepper lines were developed by conventional breeding using a jalapeno derivative and the low seed gametoclonal variant Bellsweet to develop inbred varieties that exhibited small narrow fruit that were sweet and contained few seeds. See Plant Variety Protection Certificates for Bell-Sweet (PVP 8700124) and for Vegi-Sweet (PVP 8800202); see also U.S. Pat. No. 5,066,830.
As with any valuable plant species, breeders have long used conventional cross-breeding techniques to improve existing varieties and create new cultivars. While improvements have been achieved, cross-breeding techniques are laborious and slow because of the time required to breed and grow successive plant generations.
Conventional breeding methods can only utilize those genes that are present in species that are sexually compatible with Capsicum. Thus, it would be desirable to utilize recombinant DNA technology to produce new pepper varieties and cultivars in a controlled and predictable manner that contain genes both from sexually compatible crops, and from other unrelated plants, animals, bacteria and viruses.
The recombinant DNA manipulation of pepper, however, has been hindered by difficulty in regenerating whole plants from tissue culture, by difficulty in obtaining transformed pepper tissue and ultimately, in linking regeneration with transformation.
For these reasons, it would be desirable to provide improved methods for the recombinant DNA transformation of pepper plant material and the regeneration of whole plants from the transformed material. It would be particularly desirable to be able to introduce desired characteristic(s) to such material(s) and to be able to regenerate viable pepper plantlets from the modified materials. Such methods should be capable of introducing a preselected exogenous gene(s) into the pepper plant material and should permit the selection of transformed shoots which are regenerated from the material. The method should produce regenerated pepper plants which have stably incorporated the gene(s).
Regeneration of pepper plants in the absence of transformation is a method which has several important uses. The method can be used to generate many copies of the regenerated plant (micropropagation). It can also be used to produce valuable sources of variation (e.g., somaclonal variation) which can be used in breeding strategies for pepper improvement. Somaclonal variation can be in the form of single or multiple gene changes (mutations) or in the form of polyploidization (e.g., tetraploids or aneuploids). Tetraploids often have use in breeding strategies because they may exhibit improvements in agronomically important characteristics (e.g., disease resistance and fruit size); they can be used in strategies to induce seedlessness (e.g., via crossing with diploids to produce triploids); or they can be used to facilitate gene transfer from wild relatives via conventional breeding (e.g., to improve chromosome pairing in interspecific hybrids).
Thus, it would be desirable to provide for an improved method for the regeneration of pepper plants in the absence of transformation, and particularly for a method for the regeneration of tetraploid peppers.